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Investigating the function of a member of an organelle-
dedicated RNA binding protein family
Maritza Duarte
Barkan Lab
University of Oregon
Institute of Molecular Biology
Origin of Mitochondria and Chloroplasts
Transfer of some organellar genes to the nucleus
Dual-origin of nuclear genes encoding organellar RNA binding
proteins
Co-opted host derived
Bacterial ancestorsBacterial ancestors
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* Group IIintrons
However CP. introns require nuclear encoded proteins for splicing
Many Group II introns in The Chloroplast genome
“Self splicing” Group II introns
Nuclear Encoded Proteins required for chloroplast Group II intron splicing
My project :Grew out of the discovery of wtf1
Discovery of DUF860/ WTF1
CAF1??
??
Mass Spec
Chloroplast Extract (Stroma) IP with CAF1
antibody
SDS- PAGE
WTF1 Protein Architecture
DUF860transit peptide
????
van Wijk Lab
Found:wtf1Others…
DUF 860 gene family
WTF2
The DUF860 family in rice and Arabidopsis
• the only characterized DUF860 member is a plastid splicing factor, WTF1
• WTF1 binds RNA in vitro and in vivo.
• all members predicted to localize to the mitochondria or chloroplast.
DUF860 family characteristics
My Project will investigate the function of another chloroplast targeted DUF860 protein. This protein is named wtf2 and is hypothesized to affect CP. RNA metabolism like wtf1.
Outline of approach
• Obtaining wtf 2 mutant in maize and Arabidopsis
• Analyzing morphological and molecular phenotypes to determine wtf 2 function
Reverse Genetics with the Photosynthetic Mutant Library to find
wtf mutants
PML = ~2,000 Transposon (mu) induced non- photosynthetic mutants
WTF2
1
21
Tiffany Kroeger found the wtf2 mutants by PCR screens of the PML for mu inserts with in the wtf2 gene
Testing for Phenotype linkage of wtf2
wtf2
Plant then collect tissue
Run PCR to check for insert
Link genotype to phenotype
Obtain wtf 2 mutants
WTF2
1
wtf2 lacks Chloroplast rRNAw
tf2-2
wtf2
-1
WT
WT
why
1-2
Ladd
er
28 S
18 SCP. 16SCP. 23S *
Ribosome subunitProteins RNA.
Global analysis of chloroplast RNAs in wtf2 mutants
Total RNA
Tiling Microarray of the CP genome
Hybridized microarray with wtf2 and why1
Label with fluorescence dyes
why1
why1wtf 2
wtf 2
RNA transcriptional Profile why1 vs. wtf2 mutants
PsaB-1
petG
psaj psac
Fragment #/Chromosomal Position
L
ow
in w
tf2
H
igh
in w
tf2
trnG trnD
trnL
rpl2
trnF
How are specific CP. RNAs affected?
Psaj-1 probe Pet G probe
Rpl2 probe Psac probe Psa-B probe
P tRNG 2 probe
Possible tRNA loss specific to wtf2 mutants
Mature tRNG
Does wtf2 mutation have different consequences in Dicots vs Monocots?
no cp translation
Maize wtf2 mutant
Arabidopsis wtf2 mutant
wtf2 disruption
Can Arabidopsis wtf2 mutants germinate?
Wtf2 mutant
WT
Conclusion
Future work
Acknowledgements
• SPUR • Peter O’day
And the entire Barkan lab…
Acknowledgements
Alice Barkan Susan Belcher
Tiffany Kroeger
Jana Prikryl
Margarita Rojas
Nicholas Stiffler
Kenneth Watkins
Rosalind Williams
Bobby Coalter
Martin
Sarah